Determination of Antioxidant Activity and Sun Protection Factor of Commercial Essential Oils ^†

Abstract

Aromatic plants have been used since antiquity as great potential sources of therapeutics in folk medicine and as preservatives in foods, because they contain many biologically active compounds. Among all, essential oils (EOs) are an important group of secondary metabolites that, even if not essential for plant survival, are significant for their allelopathic effects, either negative or positive, on microbes and the environment. From the chemical point of view, EOs are highly complex mixtures involving from several tens to hundreds of different types of volatile compounds, such as terpenoids, oxygenated terpenes, sesquiterpenes, and hydrocarbons. EOs have been widely used for their virucidal, bactericidal, fungicidal, anticancer, antioxidant, and antidiabetic activities, and the biological properties of EOs are strictly linked to their chemical composition. This study was carried out on the following commercial EOs: bergamot (Citrus bergamia), bitter orange (Citrus aurantium), clove (Eugenia caryophyllata), eucalyptus (Eucalyptus globulus), fennel (Foeniculum vulgare dulce), helichrysum (Helicrysum italicum), lavender (Lavandula officinalis), lemon (Citrus limon), oregano (Origanum vulgare), palmarosa (Cymbopogon martini), star anise (Illicium verum), tangerine (Citrus reticulate), tea tree (Melaleuca alternifolia), turmeric (Curcuma longa), Chinese yin yang (mix of Eucalyptus aetheroleum, Cymbopogon citratus, Caryophylli aetheroleum, Mentha piperita, Pinus sylvestris, Salvia rosmarinus, Lavandula officinalis, Foeniculum vulgare, Salvia officinalis, Illicium verum, Mentha arvensis, Abies siberica), Japanese yin yang (Mentha arvensis), and ylang ylang (Cananga odorata). The EOs were tested for the in vitro determination of antioxidant activity (DPPH assay) and of the sun protection factor (SPF) by means of UV-Vis spectrophotometry. These biological activities allowed us to evaluate their potential application as natural preservatives and active ingredients in foods, beverages, and cosmetics, as well as in galenic preparations. The results show that amongst the seventeen EOs studied, clove showed the highest antioxidant activity, with an EC50 of 0.36 µL/mL, followed by Chinese yin yang (5.35 µL/mL), oregano (11.58 µL/mL), and ylang ylang (12.71 µL/mL). Moreover, higher SPF values were recorded for bergamot (9.74), star anise (9.28), fennel (9.10), bitter orange (8.96), ylang ylang (8.41), and clove (8.26). Overall, clove and ylang ylang EOs resulted the best potential candidates as natural preservatives, as they showed the highest health-promoting values, because at the same time, they provided protection against oxidative stress and fought free radicals that may form after sun radiation exposure.

1. Introduction

Recent awareness about the environment, healthcare, and the minor usage of synthetic chemicals has led to an increased interest in natural compounds and in developing new plant-based products. Thus, the use of plant extracts and their phytoconstituents as active ingredients is a modern ecological approach in foods, beverages, and cosmetics, as well as other industrial formulations [1,2,3]. Furthermore, these products have no side effects, a broad spectrum of action combined with high efficacy, and generally low prices [1,4].

In the plant kingdom, there are 400,000 known species of both aromatic and medicinal plants, of which about 2000 species come from nearly 60 botanical families of essential-oil-bearing plants [5,6].

Aromatic and medicinal plants have been used since antiquity in many cultures for their medicinal and therapeutic advantages, offering a variety of benefits from medicinal cosmeceuticals and dietary purposes to religious use. Many studies have discussed their uses linked to their chemical composition, since these plants are sources rich in biologically active compounds, mainly phenolics and essential oils (EOs).

EOs are highly complex mixtures involving from several tens to hundreds of different types of volatile compounds, such as terpenoids, oxygenated terpenes, sesquiterpenes, and hydrocarbons. The chemical constituents are among the factors that determine the characteristic aroma, the purity, and the therapeutic value of each EO [3,5,7]. The well-known activities of EOs, virucidal, antibacterial, antifungal, anticancer, antioxidant, and antidiabetic activities, have been extensively useful in medicinal and pharmaceutical productions, in cosmetic industries as perfumery and fragrance, and in aromatherapy and food sectors as additives and preservatives [3,5,7].

In nature, EOs play very important roles in plant defense and signaling processes. For instance, they are involved in defense mechanisms against insects, herbivores, and microorganisms, including the attraction of pollinating insects and fruit-dispersing animals, water regulation, and allelopathic interactions [8].

Nowadays, large quantities of EOs are produced globally for the industries of fragrances and flavors, and cosmetics, as well as for phytomedicine and aromatherapy. Demand comes mostly from the following markets: food and beverage (35%), fragrances, cosmetics, and aromatherapy (29%), household (16%), and pharmaceutical (15%) [9].

For all these reasons, this work aimed to study different commercial EOs, through the chemical screening of protective and health-promoting compounds, in order to evaluate their potential application as natural preservatives and active ingredients in the replacement of chemical additives in foods, beverages, and cosmetics as well as in pharmaceutical formulations. In particular, we investigated seventeen commercial EOs (bergamot, bitter orange, cloves, eucalyptus, fennel, helicrysum, lavender, lemon, oregano, palmarosa, star anise, tangerine, tea tree, turmeric, Chinese yin yang, Japanese yin yang, and ylang ylang) testing two activities in vitro: the antioxidant activity and the sun protection factor (SPF).

2. Materials and Methods

2.1. Reagents and Standards

All reagents and solvents were of analytical grade unless otherwise stated. 2,2-diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma Chemical Co. (USA).

2.2. Essential Oils

The EOs of 13 plants were purchased from the following companies:

• Bergamot from Citrus bergamia (peels; origin, Italy; A&N Fascì);
• Bitter orange from Citrus aurantium (peels; origin, Ivory Coast; Essenthya);
• Clove from Eugenia caryophyllata (buds; origin, Sri Lanka; Primavera);
• Eucalyptus from Eucalyptus globulus (leaves and twigs; origin, Spain; Phoenix Pharma);
• Fennel from Foeniculum vulgare dulce (seeds; origin, Italy; Primavera);
• Helicrysum from Helicrysum italicum (flowers; origin, Italy; FresiAromi);
• Lavender from Lavandula officinalis (flowers; origin, Bulgaria; Primavera);
• Lemon from Citrus limon (peels; origin, Italy; A&N Fascì);
• Oregano from Origanum vulgare (flowering plants; origin, Spain; Primavera);
• Palmarosa from Cymbopogon martini (flowering plants; origin, India; Essenthya);
• Star anise from Illicium verum (fruits and seeds; origin, Vietnam; Primavera);
• Tangerine from Citrus reticulate (peels; origin, Italy; Oleolio)
• Tea tree from Melaleuca alternifolia (leaves and twigs; origin, Australia; Naturando);
• Turmeric from Curcuma longa (rhizomes; origin, Madagascar; Essenthya);
• Chinese yin yang constituted by a mix of EOs (Eucalyptus aetheroleum, Cymbopogon citratus, Caryophylli aetheroleum, Mentha piperita, Pinus sylvestris, Salvia rosmarinus, Lavandula officinalis, Foeniculum vulgare, Salvia officinalis, Illicium verum, Mentha arvensis, Abies siberica (origin, China; Best of Nature));
• Japanese yin yang from Mentha arvensis (whole plant; origin, Japan; Best of Nature);
• Ylang ylang from Cananga odorata (whole plant; origin, Madagascar; Essenthya).

2.3. In Vitro Antioxidant Activity Assay

The antioxidant activity of EOs was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay according to Blois’ procedure [10]. Briefly, 1.35 mL of 60 μM DPPH radical in methanol was added to different EO concentrations. The decrease in absorbance at 517 nm was continuously determined until absorbance stabilization. The radical scavenging activity percentage (%RSA) of DPPH discoloration was calculated according to the formula:

$$\%RSA=\frac{\left(A_{DPPH}-A_s\right)}{A_{DPPH}}\times 100$$

(1)

where A_S is the absorbance of the solution when the EO was added and A_DPPH is the absorbance of the DPPH solution. The extract concentration (EC) necessary to achieve 50% of radical DPPH inhibition (EC₅₀) was obtained by plotting the RSA percentage as a function of the extract concentrations and was expressed as mg/mL, as reported by Vella et al. [2].

2.4. In Vitro Sun Protection Factor Determination

The in vitro SPF was determined according to the COLIPA standards [11] by measuring the percent transmittance across the UV spectrum (ranging from 290 to 320 nm) weighted by the erythemal factors at different wavelengths, using the following equation:

$$SPF=CF\times {{\displaystyle \sum }}_{290}^{320}EE \left(\lambda \right)\times I \left(\lambda \right)\times Abs$$

(2)

where CF = correction factor (=10), EE (λ) = erythemal effect spectrum, I (λ) = solar intensity spectrum, and Abs = absorbance values of samples.

Equation (2), obtained by Mansur et al. [12], was applied to calculate the SPF using the EE (λ) × I (λ) values determined by Sayre et al. [13], as reported in

Table 1. Values of EE (λ) × I (λ) used in the SPF calculation.

Wavelength (nm)	EE (λ) × I (λ)
290	0.0150
295	0.0817
300	0.2874
305	0.3278
310	0.1864
315	0.0837
320	0.0180

.

For the determination of the SPF, 1% v/v EO solutions were prepared in ethanol, and from this stock solution, 0.1% working concentrations were obtained. The absorbance of the sample solutions was acquired with a UV-visible spectrophotometer in the range of 290–320 nm, at 5 nm intervals, using ethanol as blank [14].

3. Results and Discussion

Due to problems with chemically synthesized preservatives and the growing demand of consumers for natural food additives and cosmetic formulations, researchers have turned their attention to plant-derived natural compounds such as EOs.

In this study, the in vitro determination of antioxidant activity (DPPH assay) and of the sun protection factor (SPF) were carried out on the following seventeen commercial EOs: bergamot, bitter orange, clove, eucalyptus, fennel, helicrysum, lavender, lemon, oregano, palmarosa, star anise, tangerine, tea tree, turmeric, Chinese yin yang, Japanese yin yang, and ylang ylang.

The principle of scavenging the stable DPPH radical is extensively used to determine the antioxidant capacity of EOs. In particular, the assay is based on the ability of a potential antioxidant compound to reduce the DPPH radical, aging as a hydrogen donor.

In this study, the EOs of bergamot, cloves, fennel, helicrysum, lavender, lemon, oregano, palmarosa, star anise, tea tree, turmeric, Chinese yin yang, and ylang ylang were able to inhibit 50% of the radical scavenging activity of DPPH, as showed in

Table 2. Antioxidant activity (expressed as EC₅₀) of EOs.

Essential Oil	EC50 (µL/mL)
Bergamot	128.09 ± 0.63
Bitter orange	n.d.
Clove	0.36 ± 0.02
Eucalyptus	n.d.
Fennel	90.86 ± 0.14
Helicrysum	373.48 ± 0.52
Lavender	665.54 ± 0.50
Lemon	760.68 ± 0.77
Oregano	11.58 ± 0.22
Palmarosa	950.52 ± 0.71
Star anise	500.57 ± 0.33
Tangerine	n.d.
Tea tree	54.81 ± 0.24
Turmeric	24.99 ± 0.44
Chinese yin yang	5.35 ± 0.13
Japanese yin yang	n.d.
Ylang ylang	12.71 ± 0.17

n.d. = not detected.

. On the contrary, bitter orange, eucalyptus, tangerine, and Japanese yin yang revealed no antioxidant activity.

The results show that amongst the seventeen EOs studied, clove showed the highest antioxidant activity with an EC₅₀ of 0.36 µL/mL, followed by Chinese yin yang (5.35 µL/mL), oregano (11.58 µL/mL), and ylang ylang (12.71 µL/mL). Furthermore, turmeric displayed a moderate antioxidant activity, 24.99 µL/mL, while the remaining EOs (bergamot, fennel, helicrysum, lavender, lemon, palmarosa, star anise, and tea tree) revealed weak antioxidant activity, with values ranging from 54.81 µL/mL to 950.52 µL/mL, as reported in Table 2.

The in vitro SPF measurement represents an admissible and fast tool to narrow in vivo experiments and related risks to UV exposure. SPF determination is a useful test for screening ingredients widely employed in the food and cosmetic fields. In particular, this methodology may be useful as a rapid control tool during the production processes of food additives or supplements and cosmeceutical products and in the analysis of the final products and may give important information before proceeding to in vivo tests [14]. The higher the SPF is, the more protection is offered by phytoconstituents against UV light. In fact, EOs, if correctly mixed in food as natural preservatives and in cosmeceutical formulations, should absorb UV radiations (290–400 nm) in a such manner that confers the matrices the capability to prevent skin damage and to counteract other health problems related to free radicals formed by sun exposure [14].

In this study, the highest SPF value was recorded for bergamot, 9.74, followed by star anise (9.28), fennel (9.10), bitter orange (8.96), ylang ylang (8.41), and clove (8.26), as depicted in Figure 1.

On the other hand, helicrysum, turmeric, tangerine, and Chinese yin yang EOs showed minor SPF values, 6.91, 5.26, 3.75, and 3.02, respectively. Further, it was observed that eucalyptus, lavender, lemon, oregano, palmarosa, tea tree, and Japanese yin yang EOs possessed very low sun protection factors, around 2 or less.

Generally, the calculation of antioxidant activity and SPF may help in the selection of the best EO chemical profile, since biological activities are linked with them, and thus, their quality and application.

Moreover, the growing interest in underutilized cultivars to be devoted to the food and cosmetic markets, according to the emergent demands of new applications, could be explored by means of the routine study of their EO biological activities, i.e., antioxidant activity and SPF properties, as reported in this research study.

4. Conclusions

The increasing demand of natural phytoconstituents from EOs can be due to their reduced side effects compared with their chemical counterparts, their broad spectrum of action combined with a high efficacy, and their generally low costs.

Overall, in this study, clove and ylang ylang EOs resulted to be the most effective candidates as natural preservatives to be used as sources of health-promoting compounds, at the same time providing protection against oxidative stress and fighting free radicals that naturally tend to form with sun exposure.

It can be concluded that the combined antioxidant activity and SPF properties of EOs can provide synergistic protective effects as food additives or in cosmeceutical formulation.

EOs may be recognized and appreciated as antioxidants capable to act in the food sector as natural preservatives, thus avoiding the potential negative effects on human health of synthetic ones. Moreover, EOs may also be valuable for increasing the shelf life of foodstuffs, drinks, and cosmetics, as they can be used as antioxidant agents in order to prevent natural oxidation and deterioration.